Biomedical subjects

Paul M M Weers

Publications and source records attributed to Paul M M Weers.

10 recordsLinked to original sources

PubMed2006

Apolipophorin III: lipopolysaccharide binding requires helix bundle opening.

Apolipophorin III (apoLp-III) is a prototypical apolipoprotein used for structure-function studies. Besides its crucial role in lipid transport, apoLp-III is able to associate with fungal and bacterial membranes and stimulate cellular immune responses. We recently demonstrated binding interaction of apoLp-III of the greater wax moth, Galleria mellonella, with lipopolysaccharides (LPS). In the present study, the requirement of helix bundle opening for LPS binding interaction was investigated. Using site-directed mutagenesis, two cysteine residues were introduced in close spatial proximity (P5C/A135C). When the helix bundle was locked by disulfide bond formation, the tethered helix bundle failed to associate with LPS. In contrast, the mutant protein regained its ability to bind upon reduction with dithiothreitol. Thus, helix bundle opening is a critical event in apoLp-III binding interaction with LPS. This mechanism implies that the hydrophobic interior of the protein interacts directly with LPS, analogous to that observed for lipid interaction.

Apolipoproteins↗

PubMed2006

Tyrosine fluorescence analysis of apolipophorin III-lipopolysaccharide interaction.

Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein that binds to lipopolysaccharides (LPS). Polyacrylamide gel electrophoresis analysis demonstrated that apoLp-III from Galleria mellonella associated with various truncated LPS variants, including lipid A. Subsequent binding studies were performed employing the intrinsic tyrosine fluorescence properties of apoLp-III, which is highly quenched in the unbound state. A marked increase in tyrosine fluorescence intensity was observed upon binding to LPS or detoxified LPS, indicating a new microenvironment for Tyr-142. This also implies that the LPS carbohydrate region is involved in LPS binding. Dissociation constants (Kd) measured by apoLp-III titration were estimated at approximately 1 microM. Increasing the ionic strength did not decrease the Kd, neither did LPS phosphate removal. In addition, truncation apoLp-III mutants, lacking two complete helices, were still able to associate with LPS. This indicates that the association of apoLp-III with LPS may not be governed by charge but by hydrophobic interactions.

Animals↗

PubMed2006

Apolipophorin III: role model apolipoprotein.

It has been one-quarter century since the identification of apolipophorin III (apoLp-III) as an important component of insect hemolymph lipid transport processes. Original studies of flight-related lipid transport that led to the discovery of apoLp-III have been followed by detailed studies of its structure and function relations, species distribution as well as its physiological roles beyond lipid transport. The non-exchangeable apoLp-I and -II, which are derived from a common precursor, are structural protein components of the multifunctional lipophorin particle. ApoLp-I/II have been identified as members of a broad lipid-binding protein family based on sequence similarities with their vertebrate counterparts. By contrast, apoLp-III can be found as a lipid-free hemolymph protein that associates with lipophorin during hormone-induced lipid mobilization. Based on structural characterization, apoLp-III belongs to a large family of exchangeable apolipoproteins characterized by segments of amphipathic alpha-helix. The remarkable structural adaptability of apoLp-III can be ascribed to its globular amphipathic alpha-helix bundle conformation wherein hydrophobic lipid-binding regions are stabilized in the absence of lipid by helix-helix interactions. Upon exposure to potential lipid surface-binding sites, the globular helix bundle opens to expose its hydrophobic interior permitting substitution of helix-helix contact in the bundle for helix-lipid interactions. Novel functions of apoLp-III beyond lipid transport have been identified recently. The expanding role of apoLp-III in innate immunity promises to offer exciting research opportunities in the future.

Animals↗

PubMed2005

Role of buried polar residues in helix bundle stability and lipid binding of apolipophorin III: destabilization by threonine 31.

Apolipophorin III (apoLp-III) from Locusta migratoria is a model exchangeable apolipoprotein that plays a key role in neutral lipid transport. The protein is comprised of a bundle of five amphipathic alpha-helices, with most hydrophobic residues buried in the protein interior. The low stability of apoLp-III is thought to be crucial for lipid-induced helix bundle opening, to allow protein-lipid interactions. The presence of polar residues in the hydrophobic protein interior may facilitate this role. To test this, two buried polar residues, Thr-31 and Thr-144, were changed into alanine by site-directed mutagenesis. Secondary structure analysis and GdnHCl- and temperature-induced denaturation studies indicated an increase in alpha-helical content and protein stability for T31A apoLp-III compared to wild-type apoLp-III. In contrast, T144A had a decreased alpha-helical content and protein stability, while tryptophan fluorescence indicated increased exposure of the hydrophobic interior to buffer. Two mutant proteins that had lysine residues introduced in the hydrophobic core displayed a more pronounced decrease in secondary structure and protein stability. Lipid binding studies using phospholipid vesicles showed that T31A apoLp-III was able to transform phospholipid vesicles into discoidal particles but at a 3-fold reduced rate compared to wild-type apoLp-III. In contrast, the less stable apoLp-III mutants displayed an increased ability to transform phospholipid vesicles. These results demonstrate the inverse correlation between protein stability and the ability to transform phospholipid vesicles into discoidal protein-lipid complexes and that Thr-31 is a key determinant of the relatively low protein stability, thereby promoting apoLp-III to interact with lipid surfaces.

Amino Acid Substitution↗

PubMed2004

Lipopolysaccharide binding of an exchangeable apolipoprotein, apolipophorin III, from Galleria mellonella.

A new role of apolipophorin III (apoLp-III) as an immune activator has emerged recently. To gain insight into this novel function, the interaction of apoLp-III with lipopoly-saccharide (LPS) was investigated. ApoLp-III from Galleria mellonella was incubated with LPS from Escherichia coli O55:B5, and analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE). Protein staining showed that apoLp-III mobility was significantly reduced. In addition, silver and LPS fluorescent staining demonstrated that LPS mobility was increased upon incubation with apoLp-III. This result suggests association of apoLp-III with LPS. Sodium dodecyl sulfate (SDS) PAGE analysis showed decreased apoLp-III mobility upon LPS addition, indicative of LPS apoLp-III interaction in the presence of SDS. The unique tyrosine residue that resides in apoLp-III was used to provide additional evidence for LPS binding interaction. In the absence of LPS, apoLp-III tyrosine fluorescence was relatively low. However, LPS addition resulted in a progressive increase in the fluorescence intensity, indicating tertiary rearrangement in the environment of tyrosine 142 upon LPS interaction. Other well-characterized apoLp-IIIs were also examined for LPS binding. Manduca sexta , Bombyx mori and Locusta migratoria apoLp-III were all able to interact with LPS. The ability of apoLp-III to form complexes with LPS supports the proposed role of apoLp-III in innate immunity.

Animals↗

PubMed2003

Structure-guided protein engineering modulates helix bundle exchangeable apolipoprotein properties.

Apolipoprotein (apo) E plays a major role in lipid metabolism by mediating cellular uptake of lipoprotein particles through interaction with members of the low density lipoprotein (LDL) receptor family. The primary region of apoE responsible for receptor binding has been limited to a cluster of basic amino acids between residues 134 and 150, located in the fourth helix of the N-terminal domain globular helix bundle structure. To investigate structural and functional requirements of this "receptor binding region" we engineered an apolipoprotein chimera wherein residues 131-151 of human apoE were substituted for residues 146-166 (helix 5) of Manduca sexta apolipophorin III (apoLp-III). Recombinant hybrid apolipoprotein was expressed in Escherichia coli, isolated, and characterized. Hybrid apolipoprotein and apoE3-N-terminal, but not apoLp-III, bound to heparin-Sepharose. Far UV circular dichroism spectroscopy revealed the presence of predominantly alpha-helix secondary structure, and stability studies revealed a urea denaturation midpoint of 1.05 m, similar to wild-type apoLp-III. Hybrid apolipoprotein-induced dimyristoylphosphatidylcholine (DMPC) bilayer vesicle solubilization activity was significantly enhanced compared with either parent protein, consistent with detection of solvent-exposed hydrophobic regions on the protein in fluorescent dye binding experiments. Unlike wild-type apoLp-III.DMPC complexes, disc particles bearing the hybrid apolipoprotein competed with 125ILDL for binding to the LDL receptor on cultured human skin fibroblasts. We conclude that a hybrid apolipoprotein containing a key receptor recognition element of apoE preserves the structural integrity of the parent protein while conferring a new biological activity, illustrating the potential of helix swapping to introduce desirable biological properties into unrelated or engineered apolipoproteins.

Animals↗

PubMed2003

Apolipophorin III: a lipid-triggered molecular switch.

Apolipophorin III (apoLp-III) is a low molecular weight exchangeable apolipoprotein that plays an important role in the enhanced neutral lipid transport during insect flight. The protein exists in lipid-free and lipid-bound states. The lipid-bound state is the active form of the protein and occurs when apoLp-III associates with lipid-enriched lipophorins. ApoLp-III is well characterized in two evolutionally divergent species: Locusta migratoria and Manduca sexta. The two apolipoproteins interact in a similar manner with model phospholipid vesicles, and transform them into discoidal particles. Their low intrinsic stability in the lipid-free state likely facilitates interaction with lipid surfaces. Low solution pH also favors lipid binding interaction through increased exposure of hydrophobic surfaces on apoLp-III. While secondary structure is maintained under acidic conditions, apoLp-III tertiary structure is altered, adopting molten globule-like characteristics. In studies of apoLp-III interaction with natural lipoproteins, we found that apoLp-III is readily displaced from the surface of L. migratoria low-density lipophorin by recombinant apoLp-III proteins from either L. migratoria or M. sexta. Thus, despite important differences between these two apoLp-IIIs (amino acid sequence, presence of carbohydrate), their functional similarity is striking. This similarity is also illustrated by the recently published NMR solution structure of M. sexta apoLp-III wherein its molecular architecture closely parallels that of L. migratoria apoLp-III.

Animals↗

PubMed2003

Lipid binding ability of human apolipoprotein E N-terminal domain isoforms: correlation with protein stability?

Human apolipoprotein (apo) E exists as one of three major isoforms, E2, E3 or E4. Individuals carrying the epsilon 4 allele have an increased risk of heart disease and premature onset of Alzheimer's disease. To investigate the molecular basis for this phenomenon, the N-terminal domain of apoE3, apoE2 and apoE4 were expressed in bacteria, isolated and employed in lipid binding and stability studies. Far UV circular dichroism spectroscopy in buffer at pH 7 revealed a similar amount of alpha-helix secondary structure for the three isoforms. By contrast, differences were noted in apoE-NT isoform-specific transformation of bilayer vesicles of dimyristoylphosphatidylglycerol (DMPG) into discoidal complexes. ApoE4-NT induced transformation was most rapid, followed by apoE3-NT and apoE2-NT. To determine if differences in the rate of apoE-NT induced DMPG vesicle transformation is due to isoform-specific differences in helix bundle stability, guanidine HCl denaturation studies were conducted. The results revealed that apoE2-NT was the most stable, followed by apoE3-NT and apoE4-NT, establishing an inverse correlation between helix bundle stability and DMPG vesicle transformation rate at pH 7. When the zwitterionic dimyristoylphosphatidylcholine (DMPC) was employed as the model lipid surface, interaction of apoE-NT isoforms with the lipid substrate was slow. However, upon lowering the pH from 7 to 3, a dramatic increase in the rate of DMPC vesicle transformation rate was observed for each isoform. To evaluate if the increased DMPC vesicle transformation rates observed at low pH is due to pH-dependent alterations in helix bundle stability, guanidine HCl denaturation studies were performed. ApoE2-NT and apoE3-NT displayed increased resistance to denaturation as a function of decreasing pH, while apoE4-NT showed no change in stability. Studies with the fluorescent probe, 8-anilino-1-naphthalene sulfonic acid, indicated an increase in apoE hydrophobic surface exposure upon decreasing the pH to 3.0. Taken together, the data indicate that changes in the stability of secondary structure elements in apoE-NT isoforms are not responsible for pH-induced increases in lipid binding activity. It is likely that pH-induced disruption of inter-helical tertiary contacts may promote helix bundle conformational changes that present the hydrophobic interior of the protein to potential lipid surface binding sites.

Apolipoproteins E↗

PubMed2002

Lipid-triggered conformational switch of apolipophorin III helix bundle to an extended helix organization.

Apolipophorin III (ApoLp-III) from the Sphinx moth, Manduca sexta, is an 18kDa protein that binds reversibly to hydrophobic surfaces generated on metabolizing lipoprotein particles. It is comprised of amphipathic alpha-helices (H1-H5) organized in an up-and-down topology forming a helix bundle in the lipid-free state. Upon interaction with lipids, apoLp-III has been proposed to undergo a dramatic conformational change, involving helix bundle opening about putative hinge loops such that H1, H2 and H5 move away from H3 and H4. In the present study, we examine the relative spatial disposition of H1 and H5 on discoidal phospholipid complexes and spherical lipoproteins. Cysteine residues were engineered at position 8 in H1 and/or at position 138 in H5 in apoLp-III (which otherwise lacks Cys) yielding A8C-, A138C- and A8C/A138C-apoLp-III. Tethering of H1 and H5 by a disulfide bond between A8C and A138C abolished the ability of apoLp-III to transform phospholipid vesicles to discoidal particles, or to interact with lipoproteins, demonstrating that these helices are required to reposition during lipid interaction. Site-specific labeling of A8C/A138C-apoLp-III with N-(1-pyrene)maleimide in the lipid-free state resulted in intramolecular pyrene "excimer" fluorescence emission indicative of spatial proximity between these sites. Upon association with dimyristoylphosphatidylcholine (DMPC) discoidal complexes, the intramolecular excimer was replaced by intermolecular excimer fluorescence due to proximity between pyrene moieties on A8C and A138C in neighboring apoLp-III molecules on the discoidal particle. No excimer emission was observed in the case of pyrene-A8C-apoLp-III/DMPC or pyrene-A138C-apoLp-III/DMPC complexes. However, equimolar mixing of the two labeled single-cysteine mutants prior to disc formation resulted in excimer emission. In addition, intramolecular pyrene excimer formation was diminished upon binding of pyrene-A8C/A138C-apoLp-III to spherical lipoproteins. The data are consistent with repositioning of H1 away from H5 upon encountering a lipid surface, resulting in an extended conformation of apoLp-III that circumscribes the discoidal bilayer particle.

Animals↗

PubMed2002

Differential lipid binding of truncation mutants of Galleria mellonella apolipophorin III.

Apolipophorin III (apoLp-III) is a prototype exchangeable apolipoprotein that is amenable to structure-function studies. The protein folds as a bundle of five amphipathic alpha-helices and undergoes a dramatic conformational change upon lipid binding. Recently, we have shown that a truncation mutant of Galleria mellonella apoLp-III comprising helices 1-3 is stable in solution and able to bind to lipid surfaces [Dettloff, M., Weers, P. M. M., Niere, M., Kay, C. M., Ryan, R. O., and Wiesner, A. (2001) Biochemistry 40, 3150-3157]. To investigate the role of the C-terminal helices in apoLp-III structure and function, two additional 3-helix mutants were designed: a core fragment comprising helix (H) 2-4, and a C-terminal fragment (H3-5). Each truncation mutant retained the ability to associate spontaneously with dimyristoylphosphatidylcholine (DMPC) vesicles, transforming them into discoidal complexes. The rate of apolipoprotein-dependent DMPC vesicle transformation decreased in the order H1-3 > H2-4 > H3-5. Truncation of two helices led to a significant decrease in alpha-helical content in buffer in each case, from 86% (wild-type) to 50% (H1-3), 28% (H2-4), and 24% alpha-helical content (H3-5). On the other hand, trifluoroethanol or complexation with DMPC induced the truncation mutants to adopt a high alpha-helical structure similar to that of wild-type protein (84-100% alpha-helical structure). ApoLp-III(H1-3) and apoLp-III(H2-4), but not apoLp-III(H3-5), were able to prevent phospholipase-C-induced low density lipoprotein aggregation, indicating that interaction of the C-terminal fragment with spherical lipoprotein surfaces was impaired. As lipoprotein binding is significantly affected and DMPC transformation rates are relatively slow upon removal of N-terminal helices, the data indicate that structural elements necessary for lipid interaction reside in the N-terminal part of the protein.

Animals↗